Background The western mosquitofish ( Gambusia affinis ) is a sexually dimorphic poeciliid fish known for its worldwide biological invasion and therefore an important research model for studying invasion biology. This organism may also be used as a suitable model to explore sex chromosome evolution and reproductive development in terms of differentiation of ZW sex chromosomes, ovoviviparity, and specialization of reproductive organs. However, there is a lack of high-quality genomic data for the female G. affinis ; hence, this study aimed to generate a chromosome-level genome assembly for it. Results The chromosome-level genome assembly was constructed using Oxford nanopore sequencing, BioNano, and Hi-C technology. G. affinis genomic DNA sequences containing 217 contigs with an N50 length of 12.9 Mb and 125 scaffolds with an N50 length of 26.5 Mb were obtained by Oxford nanopore and BioNano, respectively, and the 113 scaffolds (90.4% of scaffolds containing 97.9% nucleotide bases) were assembled into 24 chromosomes (pseudo-chromosomes) by Hi-C. The Z and W chromosomes of G. affinis were identified by comparative genomic analysis of female and male G. affinis , and the mechanism of differentiation of the Z and W chromosomes was explored. Combined with transcriptome data from 6 tissues, a total of 23,997 protein-coding genes were predicted and 23,737 (98.9%) genes were functionally annotated. Conclusions The high-quality female G. affinis reference genome provides a valuable omics resource for future studies of comparative genomics and functional genomics to explore the evolution of Z and W chromosomes and the reproductive developmental biology of G. affinis .